Nucleic acid encoding an interleukin-9 receptor variant

ABSTRACT

This invention relates to the diagnosis, treatment and methods for discovery of new therapeutics for atopic asthma and related disorders based on variants of Asthma Associated Factor 2. One embodiment of the invention is a variant of AAF2 wherein codon 173 is deleted resulting in the loss of glutamine 173 from the mature protein precursor. This single amino acid deletion results in a non-functional AAF2 protein and therefore the presence of this phenotype should be associated with less evidence of atopic asthma. Correspondingly, the lack of susceptibility to an asthmatic, atopic phenotype is characterized by the loss of glutamine at codon 171 The invention includes isolated DNA molecules which are variants of the wild type sequence as well as the proteins encoded by such DNA and the use of such DNA molecules and expressed protein in the diagnosis and treatment of atopic asthma.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of copending application Ser. No.11/371,157 (filed Mar. 9, 2006; now allowed) which is divisional ofapplication Ser. No. 10/320,646 (filed Dec. 17, 2002; now U.S. Pat. No.7,056,698, issued Jun. 6, 2006) which is a divisional of applicationSer. No. 09/596,377 (filed Jun. 16, 2000; now U.S. Pat. No. 6,602,850,issued Aug. 5, 2003) which is a divisional of application Ser. No.08/980,872 (filed Dec. 1, 1997; now abandoned) which claims the benefitof Provisional Application No. 60/032,224 (filed Dec. 2, 1996) all ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention describes biologic variability in the IL-9 receptor(Asthma Associated Factor 2) (SEQ ID NO: 1) and relates these sequencevariants to susceptibility to asthma, atopic allergy, and relateddisorders. This invention also teaches methods that utilize these IL-9receptor sequence variants for the diagnosis of susceptibility orresistance to asthma and atopic allergy. In addition, methods aredescribed that use variant IL-9 receptors in the development ofpharmaceuticals for asthma which depend on the regulation of IL-9activity.

BACKGROUND OF THE INVENTION

Inflammation is a complex process in which the body's defense systemcombats foreign entities. While the battle against foreign entities maybe necessary for the body's survival, some defense systems improperlyrespond to foreign entities, even innocuous ones, as dangerous andthereby damage surrounding tissue in the ensuing battle.

Atopic allergy is a disorder where genetic background dictates theresponse to environmental stimuli. The disorder is generallycharacterized by an increased ability of lymphocytes to produce IgEantibodies in response to ubiquitous antigens. Activation of the immunesystem by these antigens also leads to allergic inflammation which mayoccur after their ingestion, penetration through the skin, or afterinhalation. When this immune activation occurs and pulmonaryinflammation ensues, this disorder is broadly characterized as asthma.Certain cells are important in this inflammatory reaction in the airwaysand they include T cells and antigen presenting cells, B cells thatproduce IgE, and mast cells/basophils that store inflammatory mediatorsand bind IgE, and eosinophils that release additional mediators. Theseinflammatory cells accumulate at the site of allergic inflammation, andthe toxic products they release contribute to the tissue destructionrelated to the disorder.

While asthma is generally defined as an inflammatory disorder of theairways, clinical symptoms arise from intermittent airflow obstruction.It is a chronic, disabling disorder that appears to be increasing inprevalence and severity.¹ It is estimated that 30-40% of the populationsuffer with atopic allergy, and 15% of children and 5% of adults in thepopulation suffer from asthma.¹ Thus, an enormous burden is placed onour health-care resources in the treatment of these disorders.

Both the diagnosis and treatment of asthma and related disorders areproblematic.¹ In particular, the assessment of inflamed lung tissue isoften difficult, and frequently the cause of the inflammation cannot bedetermined. Although atopic asthma is an ecogenetic disorder, knowledgeabout the particular variant genes has only recently been discovered.Methods to detect these genetic variations and their role ininflammation, diagnosis and prognosis remain to be determined. What isneeded in the art is the development of technology to expedite thediagnosis of atopic asthma that specifically relates to variation ingenes responsible for susceptibility/resistance to this atopic disease.

Current treatments suffer their own set of disadvantages. The maintherapeutic agents, β-agonists, reduce the symptoms, i.e., transientlyimprove pulmonary functions, but do not affect the underlyinginflammation so that lung tissue remains in jeopardy. In addition,constant use of β-agonists results in desensitization which reducestheir efficacy and safety.² The agents that can diminish the underlyinginflammation, the anti-inflammatory steroids, have their own known listof side effects that range from immunosuppression to bone loss.²

Because of the problems associated with conventional therapies,alternative treatment strategies have been evaluated.³⁶⁻³⁹ GlycophorinA,³⁷ cyclospori,³⁸ and a nona peptide fragment of L-2,³⁸ all inhibitinterleukin-2 dependent T lymphocyte proliferation.²⁸ They are, however,known to have many other effects.² For example, cyclosporin is used as aimmunosuppressant after organ transplantation. While these agents mayrepresent alternatives to steroids in the treatment of asthmatics,³⁶⁻³⁹they inhibit interleukin-2 dependent T lymphocyte proliferation andpotentially critical immune functions associated with homeostasis. Whatis needed in the art is technology to expedite the development oftherapeutics that are specifically designed to treat the cause, and notthe symptoms, of atopic asthma. These therapies represent the mostlikely way to avoid toxicity associated with nonspecific treatment. Thetherapies would selectively target a pathway, which is downstream fromimmune functions, such as IL-2 mediated T lymphocyte activation, that isnecessary for the development of asthma and which would explain theepisodic nature of the disorder and its close association with allergy.Nature demonstrates that a pathway is the appropriate target for asthmatherapy when biologic variability normally exists in the pathway andindividuals demonstrating the variability are not immunocompromised orillt except for their symptoms of atopic asthma.

Because of the difficulties related to the diagnosis and treatment ofatopic allergies including asthma, the complex pathophysiology of thesedisorders is under intensive study. While these disorders areheterogeneous and may be difficult to define because they can take manyforms, certain features are found in common among asthmatics. Examplesof such features include abnormal skin test response to allergenchallenge eosinophilia in the lung bronchial hyperresponsiveness (BHR),bronchodilator reversibility, and airflow obstruction.³⁻¹⁰ Theseexpressions of asthma related traits may be studied by quantitative orqualitative measures.

In many cases, elevated IgE levels are correlated with BHR, a heightenedbronchoconstrictor response to a variety of stimuli.^(4,6,8,9) BHR isbelieved to reflect the presence of airway inflammation,” and isconsidered a risk factor for asthma.¹¹⁻¹² BHR is accompanied bybronchial inflammation, including eosinophil infiltration into the lungand an allergic diathesis in asthmatic individuals.^(6,8,3-18)

A number of studies document a heritable component to atopicasthma.^(4,10) Family studies, however, have been difficult to interpretsince these disorders are significantly influenced by age and gender, aswell as many environmental factors such as allergens, viral infections,and pollutants.¹⁹⁻²¹ Moreover, because there is no known biochemicaldefect associated with susceptibility to these disorders, the mutantgenes and their abnormal gene products can only be recognized by theanomalous phenotypes they produce.

The functions of IL-9 and the IL-9 receptor (the IL-9 pathway) nowextend well beyond those originally recognized. While the IL-9 pathwayserves as a stimulator of T cell growth, this cytokine is also known tomediate the growth of erythroid progenitors, B cells, mast cells, andfetal thymocytes.^(22,23) The IL-9 pathway acts synergistically withIL-3 in causing mast cell activation and proliferation.²⁴ The IL-9pathway also potentiates the IL-4 induced production of IgE, IgG, andIgM by normal human B lymphocytes,²⁵ and the IL-4 induced release of IgEand IgG by murine B lymphocytes.²⁶ A role for the IL-9 pathway in themucosal inflammatory response to parasitic infection has also beendemonstrated.^(27,28)

Nevertheless, it is not known how the sequence of the IL-9 receptorspecifically correlates with atopic asthma and bronchialhyperresponsiveness. It is known that L-9 binds to a specific receptorexpressed on the surface of target cells.^(23,29,30) The receptoractually consists of two protein ‘chains: one protein chain, known asthe IL-9 receptor, binds specifically with IL-9; the other protein chainis shared in common with the IL-2 receptor.²³ In addition, a cDNAencoding the human IL-9 receptor has been cloned andsequenced^(23,29,30) This cDNA codes for a 522 amino acid protein whichexhibits significant homology to the murine IL-9 receptor. Theextracellular region of the receptor is highly conserved, with 67%homology existing between the murine and human proteins. The cytoplasmicregion of the receptor is less highly conserved. The human cytoplasmicdomain is much larger than the corresponding region of the murinereceptor.²³

The IL-9 receptor gene has also been characterized.³⁰ It is thought toexist as a single copy in the mouse genome and is composed of nine exonsand eight introns.³⁰ The human genome contains at least four IL-9receptor pseudogenes. The human IL-9 receptor gene has been mapped tothe 320 kb subtelomeric region of the sex chromosomes X and Y.²³

In spite of these studies, no variants of the IL-9 receptor gene havebeen discovered. There is, therefore, a specific need for geneticinformation on atopic allergy, asthma, bronchial hyperresponsiveness,and for elucidation of the role of IL-9 receptor in the etiology ofthese disorders. This information can be used to diagnose atopic allergyand related disorders using methods that identify genetic variants ofthis gene that are associated with these disorders. Furthermore, thereis a need for methods utilizing the L-9 receptor variants to developtherapeutics to treat these disorders.

SUMMARY OF THE INVENTION

Applicants have discovered natural variants of the human IL-9 receptor(also known as Asthma Associated Factor 2 or AAF2) and have linked thesevariants to the pathogenesis of asthma and related disorders. Thesediscoveries have led to the development of diagnostic methods, andmethods to discover pharmaceuticals for the treatment of therapeuticsfor atopic asthma. In addition, applicants have determined that the IL-9receptor is critical to a number of antigen-induced responses in mice,including bronchial hyperresponsiveness, eosinophilia and elevated cellcounts in bronchial lavage, and elevated serum total IgE. These findingstypify atopic asthma and the associated allergic inflammation.

Furthermore, applicants have determined that a G to A nucleic acidvariant occurs at position 1273 of the cDNA (SEQ ID NO: 2) whichproduces the predicted amino acid substitution of a histidine for anarginine at codon 344 of the human IL-9 receptor precursor protein. Whenthe arginine residue occurs in both alleles in one individual, it isassociated with less evidence of atopic asthma. Thus, applicants haveidentified the existence of a non-asthmatic phenotype characterized byarginine at codon 344 when it occurs in both IL-9 receptor gene productsin one individual. As an additional significant corollary, applicantshave identified the existence of susceptibility to an asthmatic, atopicphenotype characterized by a histidine at codon 344. Thus, the inventionincludes purified and isolated DNA molecules having such a sequence aswell as the proteins encoded by such DNA.

Applicants have also determined that a splice variant of the IL-9Rexists wherein the glutamine residue at position 173 of the IL-9Rprecursor protein has been deleted (SEQ ID NO: 3) (FIG. 5). Applicantshave further shown that this variant is not able to transcribe a signalthrough the Jak-Stat pathway (FIG. 15) and is unable to induce cellularproliferation upon stimulation with 1L-9 (FIG. 16); therefore,individuals with this allele would be less susceptible to atopic asthmaand related disorders.

Applicants have further determined that a variant of the IL-9R genomicDNA exists wherein nt-213, a thymine residue in intron 5 (213 ntupstream from exon 6), has been converted to a cytosine nucleotide. Itis likely that such a variation can cause an increase in the frequencyof the splice variant which removes the glutamine residue at the startof exon 6.

In addition, applicants have discovered a variant of IL-9R wherein exon8 has been deleted (SEQ ID NO: 4) which results in a change in readingframe and a premature stop codon in exon 9. Such a variant would mostlikely be prevented from transmitting a signal through the Jak-Statpathway and, therefore, individuals with this allele would also be lesssusceptible to atopic asthma and related disorders.

The biological activity of IL-9 results from its binding to the IL-9receptor and the consequent propagation of a regulatory signal inspecific cells; therefore, IL-9 functions can be interrupted by theinteraction of IL-9 antagonists with IL-9 or its receptor. Downregulation, i.e., reduction of the functions controlled by IL-9, isachieved in a number of ways. Administering antagonists that caninterrupt the binding of IL-9 to its receptor is one key mechanism, andsuch antagonists are within the claimed invention. Examples includeadministration of polypeptide products encoded by the DNA sequences of anaturally occurring soluble form of the IL-9 receptor, wherein the DNAsequences code for a polypeptide comprising exons 2 and 3 (SEQ ID NO:5): Two other variations can produce soluble forms of the IL-9R receptorwhich comprise exons 2, 3 and 4 and In one case four amino acids from adifferent reading frame in exon 5 (SEQ ID NO: 6) (FIG. 6) and in theother case there are 27 amino acids from a different reading frame inexon 5 (SEQ ID NO: 7) (FIG. 7).

Methods to identify agonists and antagonists of the IL-9 receptorpathway can be identified by assessing receptor-ligand interactionswhich are well described in the literature. These methods can be adaptedto high throughput automated assays that facilitate chemical screeningsand potential therapeutic identification. Agonists are recognized byidentifying a specific interaction with the W-9 receptor. Loss ofbinding for a putative ligand which is labeled when a 100- to 1000-foldexcess of unlabeled ligand is used is generally accepted as evidence ofspecific receptor binding. Many labels and detection schemes can be usedduring these experiments. A similar loss of binding when increasingconcentrations of test compound are added to a known ligand and receptoris also evidence for an antagonist.

Knowledge of the variant receptors provides the means to constructexpression vectors that can be used to make soluble receptor forreceptor binding assays. Mutagenesis of these soluble receptors can beused to determine which amino acid residues are critical to bind ligandand aid in the structure-based design of antagonists. Cells lackinghuman IL-9 receptor can be transiently or stably transfected withexpression vectors containing a variant receptor and used to assay forIL-9 pathway activity. These activities may be cellular proliferation,or prevention of apoptosis which have both been ascribed to the IL-9pathway. These cells can be used to identify receptor agonists andantagonists as described above.

The methods discussed above represent various effective methodsutilizing the variant forms of IL-9 receptor to develop therapeutics foratopic asthma and other related disorders.

A number of techniques have been described that may be used to diagnoseatopic asthma that recognize single nucleotide variants in the IL-9receptor including DNA sequencing, restriction fragment lengthpolymorphisms (RFLPs), allele specific oligonucleotide analyses (ASO),ligation chain reaction (LCR), chemical cleavage, and single strandedconformational polymorphism analyses (SSCP). A skilled artisan willrecognize that the use of one or more of these techniques, as well asothers in the literature, may be used to detect one or more variationsin the IL-9 receptor gene or mRNA transcript and are within the scope ofthe present invention.

Still other techniques may be used to detect amino acid variants in theIL-9 receptor including ELISAs, immunoprecipitations, Westerns, andimmunoblotting. Thus, polyclonal and monoclonal antibodies whichrecognize specifically the structure of the various forms of the IL-9receptor are also within the scope of this Invention and are usefuldiagnostic methods for describing susceptibility or resistance to atopicasthma and related disorders.

The methods discussed above represent various effective methods fordiagnosing atopic asthma and other related disorders.

Thus, applicants have provided methods that use the IL-9 receptor toidentify antagonists that are capable of regulating the interactionbetween IL-9 and its receptor. More specifically, applicants provide amethod for assaying the functions of the IL-9 receptor to identifycompounds or agents that may be administered in an amount sufficient todown-regulate either the expression or functions of the IL-9 pathway.

Having identified the role of the IL-9 pathway in atopic allergy,bronchial hyperresponsiveness and asthma, applicants also provide amethod for the diagnosis of susceptibility and resistance to atopicallergy, asthma, and related disorders.

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and, together with the description, serve to explain theprinciple of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the human IL receptor cDNA. Boxesdepict axon 2 to 9 encompassing the coding region (relative size inscale, except the 3′ untranslated part of exon 9, outlined by dashedline). Transmembrane region is encoded by exon 7, intracellular domainby exon 8 and 9, and the extracellular by exon 2 to 6. Arrows indicatepolymorphisms or aberrant splices affecting partial sequence of theexon; a) deletion of the first 5 or 29 nucleotides in axon 5; b)deletion of the first 3 nucleotides in exon 6 (codon 173); c) arg/glypolymorphism at codon 310; d) arg/his polymorphism at codon 344; e)polymorphism at codon 410+n consisting of either 8 or 9 serines;*)complete deletion of exon 3, 4 or 8.

FIG. 2: Translated cDNA sequence of the wild type IL-9R precursorprotein with Arg allele at codon 344 (nucleotides 1272-1274) and the 8Ser/4 Asn repeats starting at codon 410 (nucleotides 1470-1472). Thecorresponding peptide (SEQ ID NO: 27) is also shown.

FIG. 3: Translated cDNA sequence of the IL-9R precursor protein with theHis allele at codon 344 (nucleotides 1272-1274) and the 9 Ser/4 Asnrepeats starting at codon 410 (nucleotides 1470-1472). The correspondingpeptide (SEQ ID NO: 28) is also shown.

FIG. 4: Translated cDNA sequence of IL-9R precursor protein with thedeletion of exon 8 causing the frame shift in exon 9, the production of11 non-wild type amino acids and a premature stop codon. Thecorresponding peptide (SEQ ID NO: 30) is also shown.

FIG. 5: Translated cDNA sequence of IL-9R precursor protein with thedeletion of Glutamine at codon 173. The corresponding peptide (SEQ IDNO: 29) is also shown.

FIG. 6: Translated cDNA sequence of IL-9R precursor protein with analternate splice in exon 5 resulting in a premature stop codon and theproduction of 27 non-wild type amino acids. The corresponding peptide(SEQ ID NO: 32) is also shown.

FIG. 7: Translated cDNA sequence of IL-9R precursor protein with analternate splice in exon 5 resulting in a premature stop codon and theproduction of 4 non-wild type amino acids. The corresponding peptide(SEQ ID NO: 33) is also shown.

FIG. 8: Translated cDNA sequence of IL-9R precursor protein with thedeletion of exon 4 producing a stop codon as the first codon of exon 5.The corresponding peptide (SEQ ID NO: 31) is also shown.

FIG. 9: Table showing the association between the IL-9 receptor genotypeand atopic allergy. The Arg/Arg individuals are homozygous for the Argallele with the 8 Ser/4 Asn repeats. The Arg/His individuals areheterozygous for the Arg allele with the 8 Ser/4 Asn repeats and the Hisallele with the 9 Ser/4 Asn repeats, 9 Ser/3Asn repeats, and 10 Ser/2Asn repeats in exon 9. The His/His individuals are homozygous for theHis allele with the 9 Ser/4 Asn repeats, 9 Ser/3 Asn repeats, and 10Ser/2 Asn repeats in exon 9. The Arg/Arg individuals are protected fromatopic allergy. The Arg/His and His/His individuals are susceptible toatopic allergy (P=0.002).

FIG. 10: Map of the expression construct of the IL-9 receptor.

FIG. 11: Western blot of recombinant IL-9 receptor proteins (left: Argallele with the 8 Ser/4 Asn repeats; right: His allele with the 9 Ser/4Asn repeats) using C terminal antibody probe in TK transfected cellline.

FIG. 12: Expression of human IL9 receptor variants in TS1 cells showingdifferential mobility between the Arg 344 variant with B Ser/4 Asnrepeats (GM) and His 344 variant with 9 ser/4 Asn repeats (GH9). Amobility shift is seen demonstrating differential post-translationalmodification of these two variant form of the IL-9 receptor.

FIG. 13: XY specific amplimers for specific amplification of the IL-9receptor gene. Pseudogenes on chromosomes 9, 10, 16, or 18 are notamplified by PCR. (M is mouse DNA, H is human DNA, and C is hamsterDNA.)

FIG. 14: Immunoreactivity of an anti-human IL9 receptor neutralizingantibody with wild type and Δ-Q receptors. Panel A): COS7 cells weretransiently transfected with the LXSN vector alone (A and B), wild typeIL-9R (C and D), Wild type IL-9R with 9 Ser residues starting with codon410 (E and F), A-Q 173 variant (G and H) and Δ-Q 173 with 9 Ser residuesstarting at codon 410 (I and J) and sequentially incubated with MAB290and anti-mouse IgG Texas Red-conjugated antibody (B,D,F,H and J) asdescribed (Example 8). DAPI staining (A,C,E,G and I) was included tovisualize every cell in the photographed field. Panel B): as in A)except that cells were first fixed/permabilized and then incubated witha C-terminal specific antibody (sc698) followed by incubation withanti-rabbit IgG Texas Red-conjugated antibody. Bar=10 microns.

FIG. 15: Activation of members of Jak, Stat, and Irs families viadifferent variants of the human IL-9 receptor. TS1 cells expressingeither GH9, ΔQGR8, or ΔQGH9 were starved for 6 hours and then treatedfor 5 minutes without cytokine (−), with murine IL-9 (m), or with humanIL-9 (h). Cell extracts were immunoprecipitated with various antibodiesspecific for different members of Jak, Stat and Irs families.Immunoblots were first reacted with an anti-phosphotyrosine antibody todetect only tyrosine-phosphorylated proteins and then stripped andreprobed with the same antibody used to immunoprecipitate each protein.GH9, ΔQGR8, ΔQGH9 are as indicated in FIG. 16.

FIG. 16: Proliferation of TS1 cells expressing different forms of humanIL-9 receptor. Cells were seeded in quadruplicate in 96-well plates(1000/well) and treated without cytokine or with murine or human IL-9 (5ng/ml). A colorimetric assay was performed 7 days later to determinecell number, and the ratio between treated/untreated cells (% control)was calculated to assess growth rate. L×SN=cells transfected with theempty vector; GR8 is wild type IL-9R; GH9 is the His 344 variant with 9Ser residues starting at codon 410; ΔQGR8 and ΔAQGH9 are the ΔQ173variants on the wild type and the His 344+9—Ser background,respectively.

FIG. 17: Genomic DNA sequence of intron 5 of the IL-9R with a variationat nucleic acid 213 nt upstream from axon 6 where a T residue is changedto a C residue as indicated by the arrow.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have resolved the needs in the art by elucidating an IL-9pathway, and compositions that affect that pathway, which may be used inthe treatment, diagnosis, and development of methods to’ identify agentsto prevent or treat atopic asthma and related disorders.

Asthma encompasses inflammatory disorders of the airways with reversibleairflow obstruction. Atopic allergy refers to atopy, and relateddisorders including asthma, bronchial hyperresponsiveness (BHR),rhinitis, urticaria, allergic inflammatory disorders of the bowel, andvarious forms of eczema. Atopy is a hypersensitivity to environmentalallergens expressed as the elevation of serum total IgE or abnormal skintest responses to allergens as compared to controls. BHR refers tobronchial hyperresponsiveness, a heightened bronchoconstrictor responseto a variety of stimuli.

By analyzing the DNA of individuals that exhibit atopic allergy andasthma-related disorders, applicants have identified polymorphisms inthe IL-9 receptor (IL-9R) gene that may correlate with the expression ofasthma. The IL-9 receptor gene (also known as Asthma Associated Factor 2or AAF2) refers to the genetic locus of interleukin-9 receptor, acytokine receptor associated with a variety of functions involving theregulation of human myeloid and lymphoid systems. The human IL-9receptor gene of the present invention is found in the subtelomericregion of the XY chromosomes.

By polymorphism, applicants mean a change in a specific DNA sequence,termed a “locus,” from the prevailing sequence. In general, a locus isdefined as polymorphic when artisans have identified two or more allelesencompassing that locus and the least common allele exists at afrequency of 1% or more.

Specific amplification of the authentic IL-9R (gene encoding for thebiologically functional protein located in the XYq pseudoautosomalregion) using standard primer design was not possible because IL-9R hasfour highly homologous (>90% nucleotide identity), non-processedpseudogenes at other loci in the human genome (chromosome 9, 10, 16,18). Because of the high identity of these other genes, genomic PCRamplification using standard primer design resulted in co-amplificationof all genes, thus making sequence analysis of the authentic geneequivocal. In order to study authentic IL-9R structure as it may relateto predisposition to disease such as asthma, discussed in thisapplication, or other diseases such as cancer (Renauld, et al.,Oncogene, 9:1327-1332, 1994; Gruss, et al., Cancer Res., 52:1026-1031,1992); applicants have designed specific amplimers. The specific primerswere found to be authentic for IL-9R amplification with no amplificationof the 4 pseudogenes. The primer sequences are shown in Example 2 andtheir specificity is demonstrated in FIG. 13.

Applicants have also amplified, by RT-PCR, the entire coding region ofthe IL-9 receptor cDNA using RNA extracted from PBMCs (peripheral bloodmononuclear cells) purified from 50 donors. FIG. 1 illustrates the mostfrequent variations found in 50 Individuals analyzed. Exon 3, 4, 5, 6and 8 were affected by aberrant splicing events in samples wherefull-length cDNAs could also be cloned. Some transcripts showed completedeletion of exon 3, which causes a frameshift creating a stop codonafter a stretch of 79 unrelated residues. In the case of deletion ofexon 4, a frameshift is also generated and the first codon in exon 5 isconverted to a stop codon. In some other cDNAs, exon 5 presented partialdeletion of the first 5 or 29 nucleotides, both deletions leading toframeshifts resulting in early stop codons within exon 5. Hence, in allinstances, the putative truncated protein would lack most of theextracellular domain as well as all the transmembrane and cytoplasmicdomains. If secreted, these forms might function as soluble receptors.Finally, the first three nucleotides of exon 6, corresponding to codon173, were frequently found spliced out, resulting in deletion of theglutamine at this codon with no other changes in the remaining proteinsequence. T his splice variant is possibly related to a variant found inintron 5 of the genomic DNA (SEQ ID NO: 24) which would increase thefrequency of the splice variant (FIG. 17 and Example 12).

Applicants have also found allelic variations limited to the codingsequence of exon 9. Polymorphisms involving codon 310 and 410 have beenpreviously disclosed.^(29,30) (Kermouni, A., et al., Genomics, 371-382(1995)). Codon 310 encodes for either arginine or glycine, depending onwhether the first nucleotide at that codon is an adenine or a guanidine,respectively. At codon 410 (from hereon termed “410+n”) begins a stretchof either 8 or 9 AGC trinucleotides repeats which would be translated in8 or 9 serines, respectively.

Applicants have found a new polymorphism at codon 344. Here, the secondnucleotide is either adenine or guanidine, the two possible residuesencoded by this codon being histidine or arginine, respectively.Moreover, a correspondence between codon 344 and 410+n was observedwherein arginine at codon 344 is consistently found with 8 serines atcodon 410+n and histidine at codon 344 is found with 9 serines. Thehuman IL-9 receptor cDNA originally cloned from a human megakaryobiasticleukemic cell line, Mole, presented 9 serines at codon 410+n and, unlikeapplicants' clones, an arginine at codon 344.²⁹ Another megakaryoblasticleukemic cell line UT-7 has been reported to carry the samearginine/9-serines allele.³⁰ Applicants cloned 18 cDNAs from Mole celltine and found that 6 had 8 serines at codon 410+n and arginine at codon344. The remaining ten clones presented the published sequence.Applicants also genotyped the human acute myelogenous leukemia cell lineKG-1 and found that it was histidine/9-serines homozygous.

These DNA molecules and corresponding RNA are isolated using techniquesthat are standard in the art, such as Sambrook, et al., MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress (1985). By isolated, applicants mean that the DNA is free of atleast some of the contaminants associated with the nucleic acid orpolypeptides occurring in a natural environment.

The invention also includes the proteins encoded by these nucleic acidsequences. The invention further includes fragments of the molecules. Byfragments, applicants mean portions of the nucleic acid sequence thatmaintain the function of the full sequence. As would be known in theart, fragments result from deletions, additions, substitutions and/ormodifications.

The source of the IL-9 receptor variants of the invention is human.Alternatively, the DNA or fragment thereof may be synthesized usingmethods known in the art. It is also possible to produce the compound bygenetic engineering techniques, by constructing DNA by any acceptedtechnique, cloning the DNA in an expression vehicle and transfecting thevehicle into a cell which will express the compound. See, for example,the methods set forth in Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press (1985).

The demonstration of variant IL-9 receptor sequences which may beassociated with atopic allergy and an asthma-like phenotype, and otherswhich may be associated with the lack of an asthma-like phenotype,provides methods of diagnosing susceptibility to atopic asthma andrelated disorders. Certain variants can produce soluble receptors whichcan be used for treating these disorders.

A receptor is a soluble or membrane-bound component that recognizes andbinds to molecules, and the IL-9 receptor of the invention is thecomponent that recognizes and binds to IL-9. The functions of the IL-9receptor consist of binding to IL-9 or an IL-9-like molecule andpropagating its regulatory signal in specific cells.^(29,30,34,35) HumanIL-9 has been shown to cause phosphorylation of the IL-9 receptor itselfand the activation of proteins of the Jak-Stat pathway, Jak1, Stat1,Stat3, Stat5, and Irs2, upon binding the human IL-9 receptor (Demoulin,J-B., et al., Molecular and Cellular Biology, p. 4710-4716, September1996). Applicants have examined whether IL-9R or its variants showed anybias in the activation of these proteins and extended the analysis toJak3 and Irs1, It was determined that all of the proteins of the pathwayincluding Jak3 and Irs1 were phophoralated by IL-9R activation. It wasalso determined that IL-9R variants with changes at codons 310, 344 and410+n provided the same up-regulation as wild type IL-91R. Therefore,one aspect of the invention is therapeutics for the treatment of atopicasthma which inhibit interactions in the Jak-Stat pathway.

Unlike the wild type receptor and the other tested variants, the ΔQ173variant could not activate any proteins in the Jak-Stet pathway (FIG.15). In addition, the ΔQ173 variant could not support cellularproliferation upon IL-9 stimulation (FIG. 16). Therefore, individualswho express the ΔQ173 variant are less likely to be susceptible toatopic asthma and related disorders. One aspect of the Invention,therefore, is therapeutics that increase the expression of the ΔQ173splice variant for the treatment of atopic asthma and related disorders.

One diagnostic embodiment involves the recognition of variations in theDNA sequence of the IL-9 receptor gene or transcript. One methodinvolves the use of a nucleic acid molecule (also known as a probe)having a sequence complementary to the IL-9 receptors of the inventionunder sufficient hybridizing conditions, as would be understood by thosein the art. In one embodiment, the nucleic acid molecule will bindspecifically to the codon for Arg344 of the mature IL-9 receptorprotein, or to His344, and in another embodiment will bind to bothArg344 and to His344. In yet another embodiment, it will bind to thecodon for Gin 173. These methods may also be used to recognize othervariants of the IL-9 receptor. Another method of recognizing DNAsequence variation associated with these disorders is direct DNAsequence analysis by multiple methods well known in the art.⁴⁴ Anotherembodiment involves the detection of DNA sequence variation in the IL-9receptor gene associated with these disorders.⁴⁰⁻⁴⁴ These include thepolymerase chain reaction, restriction fragment length polymorphism(RFLP) analysis, and single-stranded conformational analysis. In apreferred embodiment, applicants provide specifically for a method torecognize, on a genetic level, the polymorphism in IL-9 receptorassociated with the His344 and Arg344 alleles using an ASO PCR. In otherembodiments, the ligation chain reaction can be used to distinguishthese alleles of IL-9 receptor genes. The present invention alsoincludes methods for the Identification of antagonists of IL-9 and itsreceptor. Antagonists are compounds that are themselves devoid ofpharmacological activity, but cause effects by preventing the action ofan agonist. To identify an antagonist of the invention, one may test forcompetitive binding with a known agonist or for down-regulation ofIL-9-like functions as described herein and in the citedliterature.^(2,22-35)

Specific assays may be used to screen for pharmaceuticals useful intreating atopic allergy based on IL-9 receptor's known role on theproliferation of T lymphocytes, IgE synthesis and release from mastcells.^(29,30,33,35) Another assay involves the ability of human IL-9receptor to specifically induce the rapid and transient tyrosinephosphorylation of multiple proteins in Mole cells.³⁴ Because thisresponse is dependent upon the expression and activation of the IL-9receptor, it represents a simple method or assay for thecharacterization of potentially valuable compounds. The tyrosinephosphorylation of Star3 transcriptional factor appears to bespecifically related to the functions of the IL-9 receptor,³⁵ and thisresponse represents a simple method or assay for the characterization ofcompounds within the invention. Still another method to characterize thefunction of the IL-9 receptor involves the use of the well known murineTS1 clone transfected with a human receptor which can be used to assesshuman IL-9 function with a cellular proliferation assay.²⁹ These methodscan be used to identify antagonists of the IL-9, receptor.

In a further embodiment, the invention includes the down-regulation ofIL-9; expression or function by administering soluble IL-9 receptormolecules that bind IL-9. Applicants and Renauld, et al.²⁹ have shownthe existence of a soluble form of the IL-9 receptor. This molecule canbe used to prevent the binding of IL-9 to cell-bound receptor and act asan antagonist of IL-9. Soluble receptors have been used to bindcytokines or other ligands to regulate their function.⁴⁵ A solublereceptor is a form of a membrane-bound receptor that occurs in solution,or outside of the membrane. Soluble receptors may occur because thesegment of the molecule which commonly associates with the membrane isabsent. This segment is commonly referred to in the art as thetransmembrane domain of the gene, or membrane-binding segment of theprotein. Thus, in one embodiment of the invention, a soluble receptormay represent a fragment or an analog of a membrane-bound receptor.

Applicants have identified three splice variants of the human IL-9receptor that result in the production of proteins that could act assoluble receptors. One splice variant resulted in the deletion of axon 4which introduced a frame-shift resulting in a stop codon as the firstcodon of exon 5. This variant would produce a peptide of about 45residues that contains an epitope reactive with antibodies that blockthe IL-9/IL-9R interaction. The other two variants contain deletions inexon 5 that will produce premature stop codons early in the axon, but,in these cases, without the deletion of exon 4. These variants wouldproduce a protein of about 100 residues also containing the epitoperecognized by blocking antibody.

Soluble IL-9 receptors may be used to screen for potential therapeutics,including antagonist useful in treating atopic asthma and relateddisorders. For example, screening for peptides and single-chainantibodies using phage display could be facilitated using a solublereceptor. Phage that bind to the soluble receptor can be isolated andthe molecule identified by affinity capture of the receptor and boundphage. In addition, compound screenings for agents useful in treatingatopic asthma and related disorders can incorporate a soluble receptorand ligand that bind in the absence of an antagonist. Detection of theligand and receptor interaction occurs because of the proximity of thesemolecules. Antagonists are recognized by inhibiting these interactions.

In addition, the invention includes pharmaceutical compositionscomprising the compounds of the invention together with apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers can be sterile liquids, such aswater and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectionable solutions. Suitable pharmaceuticalcarriers are described in Martin, E. W., Remington's PharmaceuticalScience, specifically incorporated herein by reference.

The compounds used in the method of treatment of this invention may beadministered systemically or topically, depending on such considerationsas the condition to be treated, need for site-specific treatment,quantity of drug to be administered, and similar considerations.

Topical administration may be used. Any common topical formation such asa solution, suspension, gel, ointment, or salve and the like may beemployed.

Preparation of such topical formulations are well described in the artof Pharmaceutical formulations as exemplified, for example, byRemington's Pharmaceutical Science Edition 17, Mack Publishing Company,Easton, Pa. For topical application, these compounds could also beadministered as a powder or spray, particularly in aerosol form. In apreferred embodiment, the compounds of this invention may beadministered by inhalation. For inhalation therapy, the compound may bein a solution useful for administration by metered dose inhalers, or ina form suitable for a dry powder inhaler.

The active ingredient may be administered in pharmaceutical compositionsadapted for systemic administration. As is known, if a drug is to beadministered systemically, it may be confected as a powder, pill, tabletor the like, or as a syrup or elixir for oral administration. Forintravenous, intraperitoneal or intralesional administration, thecompound will be prepared as a solution or suspension capable of beingadministered by injection. In certain cases, it may be useful toformulate these compounds in suppository form or as an extended releaseformulation for deposit under the skin or intermuscular injection.

An effective amount is that amount which will down-regulate thefunctions controlled by IL-9 receptor. A given effective amount willvary from condition to condition and in certain instances may vary withthe severity of the condition being treated and the patient'ssusceptibility to treatment. Accordingly, a given effective amount willbe best determined at the time and place through routineexperimentation. It is anticipated, however, that in the treatment ofasthma and related disorders in accordance with the present invention, aformulation containing between 0.001 and 5 percent by weight, preferablyabout 0.01 to 1%, will usually constitute a therapeutically effectiveamount. When administered systemically, an amount between 0.01 and 100mg per kg body weight per day, but preferably about 0.1 to 10 mg/kg,will affect a therapeutic result in most instances.

Applicants also provide for a method to screen for the compounds thatdown-regulate the functions controlled by the IL-9 receptor. One maydetermine whether e functions expressed by IL-9 receptor aredown-regulated using techniques standard in the art.^(29,30,34,35) In aspecific embodiment, applicants provide for a method of identifyingcompounds with functions comparable to IL-9. In one embodiment, thefunctions of IL-9 receptor may be assessed in vitro. As is known to losein the art, human IL-9 receptor activation specifically induces therapid and transient tyrosine phosphorylation of multiple proteins incells responsive to IL-9. The tyrosine phosphorylation of Stat3transcriptional factor appears to be specifically elated to the actionsof the IL-9 pathway. Another method to characterize the unction of IL-9and IL-9-like molecules depends on the “stable expression” of the L-9receptors in murine TS1 clones or TF1 clones, which do not normallyexpress human receptor. These transfectants can be used to assess humanIL-9 receptor function with a cellular proliferation assay.²⁹

The invention also includes a simple screening assay for saturable andspecific ligand binding based on cell lines that express the IL-9receptor variants.^(23,29) The IL-9 receptor is expressed in a widevariety of cell types, including K562, C616645, KG-1 transfected withthe human IL-9 receptors, B cells, T cells, mast cells, HL60, HL60-clone5, TS1 transfected with the human IL-9 receptors, 32D transfected withthe human IL-9 receptors, neutrophils, megakaryocytes (UT-7 cells),³⁰the human megakaryoblastic leukemia cell line Mo7e³⁴, TF1,²⁹macrophages, eosinophiles, fetal thymocytes, the human kidney cell line293,³⁰ and murine 320 and embryonic hippocampal progenitor celllines.^(23,29,30)

The practice of the present invention will employ the conventional termsand techniques of molecular biology, pharmacology, immunology, andbiochemistry that are within the ordinary skill of those in the art.See, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, or Ausubel, et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc.

Nonetheless, we offer the following basic background information. Thebody's genetic material, or DNA, is arranged on 46 chromosomes, whicheach comprises two arms joined by a centromere. Each chromosome isdivided into segments designated p or q. The symbol p is used toidentity the short arm of a chromosome, as measured from the centromereto the nearest telomere. The long arm of a chromosome is designated bythe symbol q. Location on a chromosome is provided by the chromosome'snumber (i.e., chromosome 5) as well as the coordinates of the p or qregion (i.e., q31-q33). In addition, the body bears the sex chromosomes,X and Y. During meiosis, the X and Y chromosomes exchange DNA sequenceinformation in areas known as the pseudoautosomal regions.

DNA, deoxyribonucleic acid, consists of two complementary strands ofnucleotides, which include the four different base compounds, adenine(A), thymine (T), cytosine (C), and guanine (G). A of one strand bondswith T of the other strand while C of one strand bonds to G of the otherto form complementary “base pairs,” each pair having one base in eachstrand. A sequential grouping of three nucleotides (a “codon”) codes forone amino acid. Thus, for example, the three nucleotides CAG code forthe amino acid Glutamine. The 20 naturally occurring amino acids, andtheir one-letter codes, are as follows: Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Asparagine or Asx B Aspartic acidCysteine Cys C Glutamine Gin Q Glutamine Acid Glu E Glutamine or Glx ZGlutamic acid Glycins Gly G Histidine His H Isoleucine Ile I Leucine LeuL Lysine Lys K Methionine Met M Phenyalanine Phe F Proline Pro P SerineSer S Threonin Thr T Tryptopan Trp W Tryosine Tyr Y Valine Val V

Amino acids comprise proteins. Amino acids may be hydrophilic, i.e.,displaying an affinity for water, or hydrophobic, i.e., having anaversion to water. Thus, the amino acids designated as G, A, V, L, I, P,F, Y, W, C and M are hydrophobic and the amino acids designated as S, Q,K, R, H, D, E, N and T are hydrophilic. In general, the hydrophilic orhydrophobic nature of amino acids affects the folding of a peptide chainand, consequently, the three-dimensional structure of a protein.

DNA is related to protein as follows:

Genomic DNA comprises all the DNA sequences found in an organism's cell.It is “transcribed” into messenger RNA (“mRNA”). Complementary DNA(“cDNA”) is a complementary copy of mRNA made by reverse transcriptionof mRNA. Unlike genomic DNA, both mRNA and cDNA contain only theprotein-encoding or polypeptide-encoding regions of the DNA, theso-called “exons.” Genomic DNA may also include “introns,” which do notencode proteins.

In fact, eukaryotic genes are discontinuous with proteins encoded bythem, consisting of exons interrupted by Introns. After transcriptioninto RNA, the introns are removed by splicing to generate the maturemessenger RNA (mRNA). The splice points between exons are typicallydetermined by consensus sequences that act as signals for the splicingprocess. Splicing consists of a deletion of the intron from the primaryRNA transcript and a joining or fusion of the ends of the remaining RNAon either side of the excised intron. Presence or absence of introns,the composition of introns, and number of introns per gene, may varyamong strains of the same species, and among species having the samebasic functional gene. Although, in most cases, introns are assumed tobe nonessential and benign, their categorization is not absolute. Forexample, an intron of one gene can represent an axon of another. In somecases, alternate or different patterns of splicing can generatedifferent proteins from the same single stretch of DNA. In fact,structural features of introns and the underlying splicing mechanismsform the basis for classification of different kinds of introns.

As to the exons, these can correspond to discrete domains or motifs as,for example, functional domains, folding regions, or structural elementsof a protein; or to short polypeptide sequences, such as reverse turns,loops, glycosylation signals and other signal sequences, or unstructuredpolypeptide linker regions. The axon modules of the presentcombinatorial method can comprise nucleic acid sequences correspondingto naturally occurring axon sequences or naturally occurring axonsequences which have been mutated (e.g., point mutations, truncations,fusions).

Returning now to the manipulation of DNA, DNA can be cut, spliced, andotherwise manipulated using “restriction enzymes” that cut DNA atcertain known sites and DNA ligases that join DNA. Such techniques arewell known to those of ordinary skill in the art, as set forth in textssuch as Sambrook, et al., Molecular Cloning: A Laboratory Manual 2d ed.;Cold Spring Harbor Laboratory Press (1985) or Ausubel, et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

DNA of a specific size and sequence can then be inserted into a“replicon,” which is any genetic element, such as a plasmid, cosmid, orvirus, that is capable of replication under its own control. A“recombinant vector” or “expression vector” is a replicon into which aDNA segment is inserted so as to allow for expression of the DNA; i.e.,production of the protein encoded by the DNA. Expression vectors may beconstructed in the laboratory, obtained from other laboratories, orpurchased from commercial sources.

The recombinant vector (known by various terms in the art) may beintroduced into a host by a process generically known as“transformation:” Transformation means the transfer of an exogenous DNAsegment by any of a number of methods, including infection, directuptake, transduction, F-mating, microinjection, or electroporation intoa host cell.

Unicellular host cells, known variously as recombinant host cells,cells, and cell culture, include bacteria, yeast, insect cells, plantcells, mammalian cells and human cells. In particularly preferredembodiments, the host cells include E. coli, Pseudomonas, Bacillus,Streptomyces, Yeast, CHO, R1-1, B-W, LH, COS-J, COS-7, BSC1, BSC40,BMT10, and S69 cells. Yeast cells especially include Saccharorhyces,Pichia, Candida, Hansenula, and Torulopsis.

As those skilled in the art recognize, the expression of the DNA segmentby the host cell requires the appropriate regulatory sequences orelements. The regulatory sequences vary according to the host cellemployed, but include, for example, in prokaryotes, a promoter,ribosomal binding site, and/or a transcription termination site. Ineukaryotes, such regulatory sequences include a promoter and/or atranscription termination site. As those in the art well recognize,expression of the polypeptide may be enhanced, i.e., increased over thestandard levels, by careful selection and placement of these regulatorysequences.

In other embodiments, promoters that may be used include the humancytomegalovirus (CMV) promoter, tetracycline-inducible promoter, simianvirus (SV40) promoter, moloney murine leukemia virus long terminalrepeat (LTR) promoter, glucocorticoid inducible murine mammary tumorvirus (MMTV) promoter, herpes thymidine kinase promoter, murine andhuman-actin promoters, HTLV1 and HIV IL-9 5′ flanking region, human andmouse IL-9 receptor 5′ flanking region, bacterial tac promoter andDrosophila heat shock protein scaffold attachment region (SAR) enhancerelements.

The DNA may be expressed as a polypeptide of any length such aspeptides, oligopeptides, and proteins. Polypeptides also includetranslational modifications such as glycosylations, acetylabons,phosphorylations, and the like.

Another molecular biologic technique of interest to the presentinvention is “linkage analysis.” Linkage analysis is an analytic methodused to identify the chromosome or chromosomal region that correlateswith a trait or disorder.⁴⁷ Chromosomes are the basic units ofinheritance on which genes are organized. In addition to genes, artisanshave identified “DNA markers” on chromosomes. DNA markers are knownsequences of DNA whose identity and sequence can be readily determined.Linkage analysis methodology has been applied to the mapping of diseasegenes, for example, genes relating to susceptibility to asthma, tospecific chromosomes.^(47,48)

Applicants wish to incorporate by reference all the references set forthabove and below.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed. It is intended that the specification and examplesbe considered exemplary only with a true scope of the invention beingindicated by the claims.

Having provided this background information, applicants now describepreferred aspects of the invention.

EXAMPLE 1

Identification of IL-9 Receptor Transcript Polymorphisms

A population of 52 individuals was ascertained randomly with respect toasthma and atopy from the Philadelphia, Pa., area. Total serum IgE wereassayed by enzyme-linked immunosorbent assay (ELISA, Genzyme, Cambridge,Mass.).

To assess the structural forms of the human IL-9 receptor cDNA, PBMCsfrom these 52 unrelated donors were isolated and cultured in thepresence of PHA and PMA (described in Example 4). Previous data fromapplicants' laboratory demonstrated the kinetics of expression for IL-9receptor message in primary cultures peak at day 6 after mitogenstimulation. Applicants cultured the cells, therefore, for 6 days atwhich time the cells were harvested and their RNA and DNA were isolatedas described in Example 5.

RNAs were reverse transcribed and amplified by PCR using primersspecific for full-length IL-9 receptor cDNA as described in Example 5.Amplification products from each individual were cloned into the TA PCRcloning vector and ten clones containing the expected inserts (asdetermined by digestion and gel electrophoresis) were sequenced in theirentirety and analyzed for structural or sequence variation.

Seven major variants were identified from the above screen. These cDNAsrepresent a codon 173 deletion, an exon 4 deletion, two separatedeletions in exon 5, an axon 8 deletion, and a full-length cDNAcontaining an ARG-to-HIS change at codon 344 of the mature protein.Additional variants exist on each of these genetic backgrounds. The Argallele is associated with 8 Ser/4 Asn repeats and 7 Ser/4 Asn repeats;the His allele is associated with 9 Ser/4 Asn repeats, 9 Ser/3 Asnrepeats, and 10 Ser/2 Asn. All of these variants are depicted in FIG. 1.

Variants were cloned into the eukaryotic expression vector pCEP4(Ciontech) which contains a CMV promoter that drives the expression ofthe cloned cDNA followed by an SV40 polyadenylation signal. The vectoralso contains a hygromycin B resistance gene which is used for selectionof eukaryotic cells containing the vector and presumably expressing thecloned cDNA under control of the CMV promoter. Recombinant plasmids wereanalyzed by sequence and those plasmids containing the correct cDNAinserts were transfected into eukaryotic recipient cells such as theSyrian hamster fibroblast TK-ts13, the human glioblastoma T98G, thehuman myeloid leukemia line TF-1 and the murine myeloid precursor cellline 32D as described in Example 3. Function was biologically assessedas a response to the IL-9 ligand in growth and/or apoptosis (Examples 7and 10).

Experiments in which the full-length IL-9 receptor cDNAs containing theARG or HIS variants were performed are the TK-ts13 hamster fibroblastsor the human T98G glioblastoma cells. Cells were transfected andanalyzed 48 hours later by Western blot and in situ staining using humanspecific carboxy terminal antibodies (Santa Cruz) (Example B). In situanalysis demonstrated that both forms of receptor appeared to beexpressed in both the hamster and human lines (FIGS. 11 and 12).Interestingly, while Western blots of both forms appeared to beexpressed at equal levels in both the human and hamster lines, adifferential migration pattern appeared between the ARG and HIS receptorforms in the TS1 cell line (FIG. 12) which suggests a differentialpost-translational modification such as glycosylation, phosphorylation,etc. This biochemical difference may be the mechanism by which thealtered function results in altered phenotype. The frequency of thevarious substitutions were used as an unbiased estimate of theprevalence of each variant in the general population. Genotype wascompared to phenotype assessed by questionnaire. A diagnosis of allergyand asthma was determined by a physician reviewing the questionnaires.Individuals homozygous for the Arg344 alleles were significantly lesslikely to demonstrate evidence for allergy and asthma when compared toheterozygotes or homozygous His344 (FIG. 9).

EXAMPLE 2

Genomic—Analysis of the IL-9 Receptor Genes.

In order to perform genomic analyses of allergic and/or asthmaticindividuals, the following strategy was designed to create PCR-specificprimers for the authentic IL-9 receptor genes located on the X/Ypseudoautosomal regions and exclude the highly conserved IL-9 receptorpseudogenes located on chromosomes 9, 10, 16, and 18. First, sequencealignments were preformed between the two published pseudogenes and thegenomic sequence of the IL-9 receptor genes. Primers were then initiallydesigned in divergent regions between the authentic genes and thepseudogenes, and then analyzed by PCR using single chromosome-specifichybrids derived from Coreill DNA Repository (Camden, N.J.). If theprimers only produce correct sized products from X and Y hybrids, theywere then optimized for robust amplification. In several cases, primersdirected to the divergent regions were not XY specific; therefore,applicants introduced additional base changes In the particular primerto increase the number of mismatches higher against the pseudogenes ascompared to the IL-9 receptor gene sequence. Table 1 contains thesequence of the primers and optimal annealing temperatures forXY-specific amplification. The specificity of these primers for XYamplification are demonstrated in FIG. 13. TABLE 1 X/Y SpecificAmplimers of IL-9 Receptor EXON SENSE PRIMER ANTISENSE PRIMER TEMP SIZE2 5′-GCA GGT GGG 5′- AGG CTT GAC 68° 300 bp GAC CCA TG -3′ ATC GGA CAAC-3′ (SEQ ID NO: 8) (SEQ ID NO: 9) 3 5′- CTG GCC TGA 5′- CTG CTT CAA 62°222 bp AGT ACT TAC C-3′ TCC TGG GGA A -3′ (SEQ ID NO: 10) (SEQ ID NO:11) 4 5′- GTG AGT TCC 5′- CAA GGC CCT 64° 335 bp CCA GGA TTG A-3′ GCTCCA AA -3′ (SEQ ID NO: 12) (SEQ ID NO: 13) 5 5′- TGG GGC TTC 5′- TAT GTAGAG 62° 259 bp AGC CTC ACA TG - TGG GGA GTC TA -3′ 3- (SEQ ID NO: 15)(SEQ ID NO: 14) 6 5′- TGT ATT CTC 5′- TGA GGT GAA 62° 337 bp GAG GGC TGAG - CAG GGG AGA A -3′ 3′ (SEQ ID NO: 17) (SEQ ID NO: 16) 7 5′- COG TGGGCC 5′-′- ACA AGG GCG 60° 262 bp CTT CAT GT 3′ GCC TTT GAT 4 (SEQ ID NO:18) (SEQ ID NO: 19) 8 5′- AGG GAC GAG 5′- CCT GCC CCC 58° 376 bp GTG GGCGGA C - CAT GTT CTT 3′ 3′ (SEQ ID NO: 21) (SEQ ID NO: 20) 9 5′- ATG CTACCT 5′- GGA CAT GAT 62° 664 bp GAG CCC TTC C - GCA TCT GGC G-3′ 3′ (SEQID NO: 23) (SEQ ID NO: 22)

These primers represent a novel method for analyzing DNA sequencevariation in these genes which can be used for diagnosis ofsusceptibility or resistance to atopic asthma and related disorders.

Using this technology, each exon was examined by DNA sequence analysesfor individuals in applicants populations to detect sequence variationin the IL-9 receptor gene.⁴⁴ Sequence polymorphisms were distinguishedfrom artifact by repeated analyses. An association of the receptorvariants with the allergic phenotypes is set forth in FIG. 9. Thesequence of the receptor alleles is set forth in FIGS. 1-8.

EXAMPLE 3

IL-9 Receptor Expression and Ligand Binding Assay

Purified recombinant IL-9 and compounds potentially resembling IL-9 instructure or function are fluorescently labeled to high specificactivity by using commercially available techniques according to thesupplier's recommendations (Pierce). Human Mo7e and murine 32D cells aregrown and resuspended at 37° C. in 0.8 ml of Dulbecco's modified Eagle'smedium supplemented with 10% (vol/vol) fetal bovine serum, 50 mM2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.25 mML-glutamine or RPMI supplemented with 10% (vol/vol) fetal bovine serum,50 mM 2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine, and1.25 mM L-glutamine, respectively. TF1.1 (TF 1 cells lacking human IL-9receptors), T98G, TK, and murine 32D cells, (Examples 7 and 10) wereused as is or after transfection with the human IL-9 receptor constructsas described below. Plasmid DNA containing one of the full-length ortruncated forms of IL-9 receptors were cloned into pCEP4 plasmid(Clontech) and purified by centrifugation through Qiagen columns(Qiagen). Plasmid DNA (50 μgrams) was added to the cells in 0.4 cmcuvettes just before electroporation. After a double electric pulse(750V/74 ohms/40 microfarads and 100 V/74 ohms/2100 microfarads), thecells are immediately diluted in fresh medium supplemented with IL-9.

Stable transfected cells were generated after 14 days of selection withhygromycin B (400 μg/ml to 1.6 mg/ml). Hygromycin-resistant clones wereanalyzed for IL-9 receptor expression by Western blots and in situstaining as described in Example 8.

Cellular receptor binding is visualized directly in real time withfluorescence microscopy. Binding and internalization is followed overtime in control cells (not transfected), and with cells transfected witheach of the known IL-9 receptor variants. An excess of unlabeled ligandor blocking antibody is used in parallel experiments to demonstratespecific binding.

Soluble IL-9 receptor including amino acids 44 to 270 with or without aHA ditag is also incubated with different forms of human labeledrecombinant IL-9. Varying amounts of FLAG-tagged (1131) ligand areincubated in PBS at, room temperature for 30 minutes with 0.5 μg ofsoluble receptor. EBC buffer (50 mM Tris pH 7.5; 0.1 M NaCl; 0.5% NP40)is added (300 μl) along with 1 μg of anti-HA antibody or anti-FLAGmonoclonal antibody (IBI) and incubated for 1 hour on ice. Fortymicroliters of protein A sepharose solution were added to each sampleand mixed for 1 hour at 4° C. Samples were centrifuged for 1 minute11,000×G and pellets were washed 4 times with 500 μl of EBC. Pelletswere dissolved in 26 pi of 2×SDS buffer, boiled for 4 minutes, andelectrophoresed through an 18% SOS polyacrylamide gel. Western blotswere probed with an anti-IL-9 receptor antibody (Santa Cruz Inc.) oranti-FLAG monoclonal antibody (IBI) against FLAG-tagged rh1L-9.Therapeutic candidates are assessed by measuring the antagonism ofligand binding. Receptor expression is shown in FIGS. 11 and 14.

EXAMPLE 4

Cell Isolation and Culture

Human peripheral blood mononuclear cells (PBMC) were isolated fromhealthy donors by density gradient centrifugation using endotoxin testedFicoll-Paque PLUS according to the manufacturer (Phermacia Biotech, ABUppsala Sweden). PBMC (5×10⁶), mouse spleen cells (5×10⁶), or 5×10⁸ Molecells were cultured in 7 ml of RPMI-1640 (Bethesda Research Labs (BRL),Bethesda, Md.) supplemented to a final concentration of 10% with eitherisogenic human serum or heat-inactivated FBS. Cells were cultured for 24hm at 37° C. either unstimulated, or stimulated with either PMA 5 Ng/mVPHA 5 μg/ml, or PHA 5 μg/ml and rhIL2 50 ng/ml (R&D Systems,Minneapolis, Minn.).

EXAMPLE 5

DNA & RNA Isolations, rtPCR, Cloning, and Sequencing of PCR Products

Cytoplasmic RNA and genomic DNA were extracted after 6 days of mitogenstimulation from cultured PBMCs, as described by Nicolaides andStoeckert.⁴⁶ One μg of RNA from each source was denatured for >10minutes at 70° C. and then reverse transcribed(V+) into cDNA using 2.5units of Superscript II reverse transcriptase. (GIBCO, BRL), 1U/I RNAseInhibitor, 2.5 mM oligo d(T)16 primer, 1 mM each of dATP, dCTP, dGTP,dTTP, 50 mM KCl, 10 mM Tris-HCL, pH 7.0, 25 mM MgCl₂ at 37° C. for onehour. A mock reverse transcription reaction was used as a negativecontrol.

One-twentieth of the rt reaction was used in PCR (50 μl) containing 6.7mM MgCl₂, 16.6 mM (NH4)₂SO₄, 67 mM Tris-HCL, pH 8.8, 10 mM2-mercaptoethanol, 6% DMSO, 1.25 mM of each dNTP, 2.5U Amplitaq DNApolymerase, and 300 ng of each of the oligonucleotides representinghuman cDNA IL-9 exon 2 (forward 5′-GCT GGA CCT TGG AGA GTG-3′) (SEQ IDNO: 25) and exon 9 (reverse 5′-GTC TCA GAC AAG GGC TCC AG-3′) (SEQ IDNO: 26). The reaction mixture was subjected to the following PCRconditions: 120 seconds at 95° C., then 35 cycles at: 30 seconds at 94°C.; 90 seconds at 58° C.; 90 seconds at 72° C. Finally, the reactionmixture was cycled one time for 15 minutes at 72° C. for extension.

PCR products representing hIL-9 receptor cDNA were subjected to gelelectrophoresis through 1.5% agarose gels and visualized using ethidiumbromide staining. Products of a mock reverse transcriptate reaction, inwhich H₂O was substituted for RNA, ware used as a negative controlamplification in all experiments.

PCR products were subcloned into the TA Cloning vector (Invitrogen, SanDiego, Calif.). Amplification of the human cDNA gave a 1614 by product.Plasmids containing hIL-9 receptor cDNA inserts were isolated byconventional techniques (Sambrook, J., et al. (1989) Molecular Cloning:A Laboratory Manual Cold Spring Harbor Laboratory Press, New York).After amplification the DNA sequence including and surrounding eachinsert was analyzed by sequencing (Sanger et al., 1977, Proc. Nati.Acad. Sci. USA 74:5463) using fluorescent dideoxyterminators and on anautomated sequencer (ABI 377, Perkin Elmer) for determination ofPCR-induced or cloning-induced errors. hIL9 receptor cDNA insertswithout cloning and/or polymerase-induced sequence errors were subclonedinto expression vectors.

EXAMPLE 6 Cloning and Expression of IL-9 Receptor Constructs In Vitro

hIL-9 receptors were subcloned into the episomal eukaryotic expressionvector pCEP4 nlontech). Inserts were cloned as BamH1-Xhol fragments intothe pCEP4 polylinker in the sense orientation to the CMV promoter usingstandard techniques (FIG. 10). Constructs were expressed in cellularhosts as described.

EXAMPLE 7

Cellular Assays Using (Mo7e, 32D, TF1.1 TK ts13, and T98G

Cell lines were used to assess the function of variant IL-9 receptorsand for the screening of compounds potentially useful in the treatmentof atopic asthma. Compounds were tested for their ability to antagonizethe anti-apoptotic or baseline proliferative response elicited by IL-9.Once a baseline anti-apoptotic or proliferative response was establishedin a given cell line, a statistically significant loss of response inassays repeated three times in triplicate was considered evidence forantagonism. A true antagonistic response was differentiated fromcellular toxicity by direct observation, trypan blue staining (atechnique well known to one of normal skill in the art), or loss of acidphosphatase activity. Specificity of antagonism is assessed for eachcompound by evaluating whether the activity is demonstrated againstother proliferative agents such as interleukin 3 or interleukin 4.

Recombinant IL-9 and compounds potentially resembling IL-9 in structureor function were purified and prepared for use in the appropriate media.Putative agonists and antagonists were prepared in water, saline, orDMSO and water. Cells were used as is or after transfection with theIL-9 receptor constructs as described in Example 3. After 24 hrs ofdeprivation from growth factors, the cells were incubated without(control) or with variable amounts of purified IL-9 and compoundspotentially resembling IL-9 in structure or function.

Cell proliferation was assayed using the Abacus Cell Proliferation Kit(Clontech, Palo Alto, Calif.) which determines the amount ofintracellular acid phosphatase present as an indication of cell number.The substrate p-nitrophenol phosphate (pNPP) was converted by acidphosphatase to p-nitrophenol, which was measured as an indicator ofenzyme concentration. pNPP was added to each well and incubated at 37°C. for one hour. 1 N sodium hydroxide was then added to stop theenzymatic reaction, and the amount of p-nitrophenol was quantified usinga Dynatech 2000 plate reader (Dynatech Laboratories, Chantilly, Va.) at410 nm wavelength. Standard curves that compare cell number with opticalabsorbance were used to determine the linear range of the assay. Assayresults were only used when absorbance measurements are within thelinear range of the assay. Briefly, the assays were run withquadruplicate samples of cells in flat-bottom μtiter plates (150 or 200microliter wells) With or without ligand for 72 to 96 hours at 37degrees C. Acid phosphatase was used as a measure of the number of cellspresent. All experiments are repeated at least-twice.

Apoptosis was assayed using the Annexin V kit as described by thesupplier (Clontech) which determines dexamethesone-induced apoptosis byrecognizing extracellular phosphatidyiserine, an early marker forapoptosis. Apoptotic cell number was scored by fluorescence microscopyas a percentage of Annexin V stained cells.

The Mo7e line is a human megakaryoblastic cell line, cultured in RPMI1640 (GIBCO/BRL, Gaithersburg, Md.), 20% Fetal Bovine Serum (Hyclone)and 10 ng/ml IL-3 (R&D Systems, Minneapolis, Minn.). The T98G line is ahuman glioblastoma cell line grown in RPMI 1640 (GIBCO/BRL). The hamsterfibroblast TK-ts13 line was also used as well as the murine 32D cellline, a murine myeloid precursor line, and both were cultured in RPMI1640 (GIBCO/BRL) containing 10% fetal bovine serum (Hyclone) in addition1 ng/ml m IL-3 was used with the 32 D cell lines. TF1.1 is a humanmyeloid leukemia line known to express the IL-2 receptor gamma subunit(confirmed by Western blots and rtPCR), but, in comparison to itspredecessor (TF1), it no longer bears IL-9 receptor by rtPCR,immunostaining, and Western blot analyses. TF1.1 is cultured in RPMI1640 (GIBCO/BRL) and 10% Fetal bovine serum (Hyclone). All the celllines respond to multiple cytokines including IL-9. The cell lines werefed and reseeded at 2×10⁵ cells/ml every 72 hours.

The cells were centrifuged for 10 minutes at 2000 rpm and resuspended inRPMI 1640 with 0.5% Bovine Serum Albumin (GIBCO/BRL, Gaithersburg, Md.)and were counted using a hemocytometer and diluted to a concentration of1×10⁵ cells/ml and plated in a 96-well microtiter plate. Each wellcontained 0.15 or 0.2 ml, giving a final concentration of 10 to 50thousand cells per well depending on the cell. Mole cells werestimulated with 50 ng/ml Stem Cell Factor (SCF) (R&D Systems,Minneapolis, Minn.) alone, 50 ng/ml SCF plus 50 ng/ml IL-3 (R&D Systems,Minneapolis, Minn.), or 50 ng/ml SCF plus 50 ng/ml IL-9. A control wasincluded which contained cells and basal media only, Serial dilutions oftest compounds (i.e., recombinant IL-9 proteins, peptides, smallmolecules) were added to each test condition in triplicate. TF1.1 cellsthat were not transfected with IL-9 receptors were used as anindependent control for response and nonspecific cytotoxicity. Cultureswere incubated for 72-96 hours at 37° C. in 5% CO₂.

EXAMPLE 8

In Situ & Western Analysis of Exogenous IL-9 Receptor in TransfectedCell Lines

In situ staining of the IL-9 receptor was carried out as follows. Cellswere grown on coverslips for 24 hours and then coverslips containing theadherent cells were washed twice in phosphate buffered saline solutioncontaining calcium and magnesium(PBS) (Gibco/BRL). For Intracellularstaining of the IL-9 receptor, the cells were fixed in 4%paraformaldehyde/PBS plus 0.1% triton-X for 15 minutes at roomtemperature before treatment with anti-human IL-9 receptor antibody; forextracellular staining, cells were treated with antibody beforefixation. The cells were then washed twice in PBS and blocked with7.5°/a BSA i n dH₂O for 30 minutes at room temperature. PBS washed cellswere then incubated with a 10 μg/ml solution of anti-human IL-9 receptor(polyclonal antibody directed against the carboxy terminus of the IL-9receptor) in 1% BSA/PBS for 1 hour at room temperature. Cells werewashed three times in PBS and then incubated in 10 μg/ml solution of ananti-rabbit rhodamine-conjugated antibody in 1% BSA/PBS for 30 minutesat room temperature. Cells were then washed three times in PBS andcounter-stained using 1 μg/ml DAPI for 1 minute at room temperature.Cells were washed three times in dH₂O and fixed to a microscope slideand analyzed by fluorescence microscopy. The results for the transferredCOST cells are shown in FIG. 14.

Western blots were performed on protein lysates obtained from directlysis of cell extracts in 0.5% lysis buffer (Tris 50 mM, NaCl 150 mM,NP40), 1 mM DTT and protease inhibitors) and boiled for 5 minutes,Samples were electrophoresed on 4-20% tris-glycine SDS gels (Novex) intris-glycine running buffer. Proteins were then transferred tonitrocellulose by electroblot using the Trans Blot II apparatus (BioRed). After transfer, the membrane was blocked in TBS-T ((20 mM Tris,137 mM NaCl, pH 7.6) plus 0.05% Tween 20) plus 5% blotto for 1 hour roomtemperature. Blots were then probed using a polyclonal antibody directedto the carboxy terminus of the IL-9 receptor (1 μg/ml) in TBS-T for 1hour. Blots were then washed three times in TBS-T for 10 minutes andprobed using a secondary anti-rabbit-horse radish peroxidase conjugatedantibody (1:10,000) in TBS-T for 30 minutes. Blots were washed as aboveand then incubated with Luminol/enhancer solution (Pierce), achemiluminescent substrate, for 5 minutes at room temperature and thenexposed to film for 1-60 seconds. See FIGS. 11 and 12.

EXAMPLE 9

Methods for the Authentic IL-9R Genomic Amplification

Specific amplification of the authentic IL-9R (gene encoding for thebiologically functional protein located in the XYq pseudoautosomalregion) using standard primer design was not possible because IL-9R hasfour highly homologous (>90% nucleotide identity), nonprooessedpseudogenes at other loci in the human genome (chromosome 9, 10, 16,18). Because of the high identity of these other genes, genomic PCRamplification using standard primer design resulted in co-amplificationof all genes, thus making sequence analysis of the authentic geneequivocal. In order to study authentic IL-9R structure as it may relateto predisposition to disease such as asthma, discussed in thisapplication, or other diseases such as cancer (Renauld, et al.,Oncogene, 9:1327-1332,1 994; Gruss, et al., Cancer Res52:1026-1031,1992), specific amplimers were designed as follows:

Sequences of the IL-9R pseudogene and authentic genes were aligned usingMac Vector software. Intronic sequences surrounding each exon were theninspected for regions of diversity between the authentic gene andpseudogenes. Primers were then designed against these regions, and usedto PCR amplify human/rodent hybrid DNAs containing individual humanchromosomes. Products were run on 3% agarose gels and analyzed forauthentic IL-9R amplification with no amplification of the 4pseudogenes. Specific PCR amplification conditions were also optimizedby varying annealing temperature and buffer conditions (DMSO content5-10%). In cases where amplification of pseudogenes still occurred,nucleotide changes were entered into primer sequences to cause greaterdivergence from the pseudogenes as compared to the authentic gene,Primer sequences are shown In Example 2 and their specificity isdemonstrated in FIG. 13.

EXAMPLE 10

Cell Proliferation Assay and Cytokine Stimulation

To determine growth response of TS1 cells expressing various forms ofhuman IL-9 receptor, cells were washed with PBS and resuspended inD-MEM, 10% fetal bovine serum. 10³ cells per well were seeded intriplicate in 96-well microplates and, where appropriate, recombinanthuman IL-9 or marine IL-9 (R&D Systems, Minneapolis, Minn.) was added ata final concentration of 5 ng/ml. Cell proliferation was evaluated after7 days using an acid phosphatase assay. Briefly, 50 μl of a buffercontaining 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100 and 10 mMp-nitrophenyl phosphate (Sigma 104 phosphatase substrate) was added perwell, The plate was incubated for 1½ hours at room temperature, thereaction stopped with 10 μl/well of 1 N sodium hydroxide and theabsorbance was read on a Dynatech Model MR600 at 410 nm, To analyzetyrosine-phosphorylation of proteins of the signal transduction cascadeupon cytokine stimulation, TS1 cells expressing various forms of humanIL-9 receptor were washed with PBS, resuspended in D-MEM, 0.5% bovineserum albumin, and incubated for 6 hours at 37° C. Successively, 20×¹⁰⁸cells were treated for 5 minutes with either human IL-9 or murine IL-9(100 ng/ml) and immediately washed in cold PBS. Cells were lysed in RIPAbuffer as described in Example 11.

EXAMPLE 11

Immunoprecipitations, Immunoblotting and Antibodies

Typically, 20-50×10⁶ cells were lysed in 1 ml of RIPA buffer (PBScontaining 1% NP40, 0.5% sodium deoxycholate, 0.1% SIDS, 1 mM PMSF, 50mM sodium fluoride, 1 nM sodium orthovanadate, and 1× “Complete”protease inhibitors mixture, Cat. No. 1697498 Boehringer Mannheim) andIncubated for 45 minutes on ice. Lysates were centrifuged for 20 minutesIn an Eppendorf microcentrifuge and the supernatant recovered andtransferred to a fresh tube. For Immunopreeipkations, 1-5 μg of theantibody were added to the lysate and incubated overnight at 4° C. 20 mlof Protein A+G agarose-conjugated beads were added for 2 hrs followed byfour washings using RIPA buffer: Beads were resuspended in Laemmlibuffer and boiled for 3 minutes before electrophoresis. Proteins weretransferred onto Immobiiion-P membrane (Millipore) and detected using ahorseradish peroxidase-conjugated secondary antibody followed by achemiluminescence detection assay (Pierce). Specific antibodies formurine and human IL-9 receptor (sc698), murine Jak1, irs1, Irs2, Stat1,Stat2, Stat3, Stat4, Stat5, and phosphotyrosine (PY) were purchased fromSanta Cruz (Santa Cruz, Calif.). Anti-Jak3 and monoclonal anti-humanIL-9 receptor MAS290 were purchased from Upstate Biotechnology and R&DSystems, respectively. FIG. 15 demonstrates the activation of members ofthe Jak family via different variants of the human IL-9 receptor.

EXAMPLE 12

Identification of IL-9 Receptor Genomic Polymorphisms

Genomic DNAs were isolated from PBMCs of volunteer donors as described(Nicolaides and Stoecker, Biotechniques 8:154-156, 1990). Sequenceanalysis of intron 5 of the human IL-9R gene was performed by PCR usingprimers of sequence ID NO 14 and sequence ID NO 17 which resulted in aproduct with the approximated molecular size of 1243 basepairs,Amplifications were carried out at 94° C. for 30 seconds, 62° C. for 1.5minutes, 72° C. for 1.5 minutes for 35 cycles in buffers describedpreviously (Nicholaides et al., Genomics 30:195-206,1995). Products werethen purified and sequenced using a standard sequence protocol.Inspection of the sequences from intron 5 in multiple individuals founda nucleotide change at −213 nt upstream of axon 6 sequences whichresulted in a thymidine (published sequence) to a cytosine nucleotidechange. An example of this change is shown in FIG. 17.

While the invention has been described and illustrated herein byreferences to various specific materials, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

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1. An isolated DNA molecule having a nucleotide sequence encoding humaninterleukin-9 receptor selected from the group consisting of a sequencecontaining an G to A nucleic acid variant at position 1273, a sequencewherein nucleic acids 759-761 (SEQ ID NO: 3) have been deleted, asequence wherein nucleic acid 613-617 (SEQ ID NO: 7) have been deleted,a sequence containing a stop codon at nucleic acids 435-437 (SEQ ID NO:5), a sequence wherein nucleic acids 613-641 (SEQ ID NO: 6) have beendeleted, and fragments thereof.
 2. The isolated DNA molecule of claim 1,wherein the sequence contains an G to A nucleic acid variant at position1273 or fragments thereof.
 3. The isolated DNA molecular of claim 1,wherein nucleic acids 759-761 (SEQ ID NO: 3) have been deleted orfragments thereof. 4-15. (canceled)
 16. An isolated protein moleculehaving an amino acid sequence encoding human interleukin-9 receptorselected from the group consisting of a sequence containing a Histidineat position 344, a sequence wherein Glutamine 173 has been deleted, asequence wherein the molecule is terminated after amino acid 64, asequence wherein the molecule is encoded by the DNA of claim 4, asequence wherein the molecule is encoded by the DNA of claim 5 orfragments thereof. 17-21. (canceled)
 22. A method for detecting ordiagnosing susceptibility to asthma or a related disorder in a humansubject comprising determining the presence or absence of the DNA ofclaim 2 wherein the presence of only said DNA indicates greatersusceptibility to asthma or related disorders.
 23. A method fordetecting or diagnosing susceptibility to asthma or a related disorderin a human subject comprising determining the presence or absence of theDNA of claim 3 wherein the presence of only said DNA indicates lesssusceptibility to asthma or related disorders. 24-28. (canceled)
 29. Amethod of identifying antagonists of the IL-9 pathway comprising thesteps of: a) obtaining cells expressing the IL-9 receptor; b) contactingsaid cells with varying ratios of a mixture of IL-9 and the putativeantagonist; c) measuring the magnitude of the phosphorylation ofproteins of the Jak-Stat pathway, wherein decreased phosphorylationindicates the presences of IL-9 antagonists. 30-55. (canceled)